P990: NF-ΚB AND JAK/STAT PATHWAYS INHIBITION REVERSES FIBROSIS IN A MURINE MODEL OF INFLAMMATION-DRIVEN MYELOFIBROSIS
Authors: Ernesto José Cuenca Zamora, Pedro Jesús Guijarro Carrillo, María José López Poveda, María Luz Morales Fernández, Maria L Lozano, Rocío González-Conejero, Constantino Martínez, Raúl Teruel Montoya, Francisca Ferrer-Marín
Journal: HemaSphere (2023)

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: In myeloproliferative neoplasms (MPN), both malignant and non-malignant clones contribute to inflammatory cytokine production mediated by JAK/STAT activation. Available JAK signaling-only inhibitors, however, do not reverse medullary fibrosis. As the NF-κB pathway also plays a critical role, we previously demonstrated the importance, in MPN, of miR-146a, a negative regulator of the NF-kB pathway by inhibiting IRAK1/TRAF6 (Leukemia 2020). Indeed, miR-146a-/- (KO) mice develop a myelofibrosis (MF)-like phenotype and show an increase in p-STAT3 with aging. Aims: To assess whether inhibition of NF-κB and/or JAK/STAT signaling can attenuate the inflammatory state and reverse the MF-like phenotype in an aged miR-146a-/- mouse model, using either (a) the JAK1/2 inhibitor ruxolitinib (RUX), (b) the NF-κB pathway inhibitor, through IKKα/β, BMS-345541 (BMS), (c) RUX+BMS, or (d) the dual JAK2/IRAK1 inhibitor pacritinib (PAC). Methods: 11-month-old miR-146a-/- (KO) mice were fed orally ad libitum for 1 month with either RUX 75 mg/kg/day, BMS 100 mg/kg/day, RUX+BMS at the same concentrations, or PAC 150 mg/kg/day. Untreated KO and wild-type (WT) mice of the same age were used as controls (n=7-15 mice/group). The human SET2 cell line was used as a JAK2V617F+/- in vitro model for pSTAT3, IRAK1 and nuclear p-p65 by western blot (WB). Results: As expected, KO (vs. WT) mice exhibited cytopenias, increased plasma levels of IL6, IL1β and TNFα (by Luminex), severe reticulin fibrosis with osteosclerosis in Bone Marrow (BM) (by reticulin staining), and large spleen with architecture loss and foci of extramedullary hematopoiesis (Figure). Consistent with histology, at the cellular level, KO mice showed significantly increased myeloid (CD11b+) and decreased lymphoid (CD19+) populations in the spleen. Accordingly, we observed drastically reduced levels of pSTAT3, STAT3, STAT5, IRAK1 and p65 in total spleen lysates. NF-κB pathway inhibitors (BMS and PAC) improved platelet counts, but only PAC also improved monocyte counts without worsening anemia; RUX did not improve monocyte or platelet counts, and the RUX+BMS combination worsened anemia. Regarding proinflammatory cytokines, plasma IL6 levels decreased significantly with RUX; IL1β levels decreased with all NF-κB inhibitors but not with RUX, although the decrease was significant only with BMS; and TNFα levels decreased significantly with all treatments. Despite all treatments reduced spleen size and partially restored its architecture (Figure), only PAC and BMS (NF-κB inhibitors) significantly reduced the number of splenic megakaryocytes. In line with the recovery of splenic architecture, NF-κB inhibitors (but not RUX) achieved a partial but significant recovery of STAT5, IRAK1 and p65 levels in total spleen lysates, as seen in WT mice. Although NF-κB inhibitors [BMS (+/-RUX) and PAC], unlike RUX, reduced severe reticulin fibrosis and osteosclerosis in BM, RUX+BMS combination induced BM aplasia (Figure), consistent with the worsening anemia seen with this treatment. Finally, in SET2 cells, RUX or BMS monotherapy inhibited JAK/STAT or NF-κB signaling respectively, but only PAC and RUX+BMS were able to inhibit both pathways. Summary/Conclusion: In an aging-associated MF-like murine model without driver mutations, NF-κB inhibitors, either alone or in combination with JAK inhibitors, as well as dual-acting inhibitors, reduced inflammatory cytokines and splenomegaly, reversed thrombocytopenia and improved BM fibrosis. The combination therapy (RUX+BMS) produced BM aplasia and worsened anemia, whereas the dual inhibitor (PAC) improved or stabilized hematologic parameters.Keywords: Bone Marrow Fibrosis, Myelofibrosis, NF- B, Myeloproliferative disorder